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First cyclotide from Hybanthus (Violaceae)
Phytochemistry 58 (2001) 47–51 www.elsevier.com/locate/phytochem
First cyclotide from Hybanthus (Violaceae) Adriana M. Broussalisa,1, Ulf Go¨ranssonb,1, Jorge D. Coussioa, Graciela Ferraroa, Virginia Martinoa, Per Claesonb,* a
Instituto de Quı´mica y Metabolismo del Fa´rmaco (IQUIMEFA) (UBA-CONICET), Ca´tedra de Farmacognosia, Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires, Argentina b Division of Pharmacognosy, Department of Medicinal Chemistry, Uppsala University, Biomedical Centre, Box 574, SE-751 23 Uppsala, Sweden Received 26 January 2001; received in revised form 23 March 2001
Abstract Hypa A, a novel macrocyclic polypeptide containing 30 amino acid residues, has been isolated from the n-butanol extract of the Argentine plant Hybanthus parviflorus. The sequence, cyclo-(SCVYIPCTITALLGCSCKNKVCYNGIPCAE), was determined by automated Edman degradation, quantitative amino acid analysis and nanospray MS/MS2. Three intramolecular disulfide bridges stabilize the cyclic peptide backbone of hypa A. Using these structural features to classify the peptide as a cyclotide, we extended the distribution of that substance class to a new genus, and now propose a uniform nomenclature for cyclotides. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Hybanthus parviflorus (Mutis ex L. F.) Baill.; Violaceae; Violetilla; Isolation; Primary structure; Cyclotides; Macrocyclic polypeptides; Hypa A; LCQ
1. Introduction The generic name ‘cyclotides’ (cyclic peptides) has been given to a rapidly emerging family of macrocyclic polypeptides, which are approximately 30 amino acid residues in length and are cyclized via the amide bonds of the peptide backbone (Craik et al., 1999). Their known taxonomic distribution has been limited to genera of Violaceae and Rubiaceae (Table 1). Recently, the characteristic cyclic cystine knot has also been found in peptides isolated from the family Cucurbitaceae (Hernandez et al., 2000). To further investigate the occurrence of cyclotides within Violaceae, we examined Hybanthus parviflorus (Mutis ex L. F.) Baill. H. parviflorus is a perennial shrub, widely distributed in the tropical and subtropical regions of America from Colombia to Argentina (Correa, 1988). In annual cultivations, it is a weed that grows on stubble, walls, rubble and wasteland (Zuloaga and Morrone, 1999). In several South American countries, including Colombia, Chile, * Corresponding author. Tel.: +46-18-471-44-79; fax: +46-18-50-9101. E-mail address: [email protected] (P. Claeson). 1 These authors contributed equally to this work.
Peru and Argentina, it is used as an emetic and purgative drug, and has substituted for ipecacuanha in medicine (Soukup, 1986; Murillo, 1889; Rutter, 1990; Garcı´a Barriga, 1992; Mazorca, 1997). In Argentina, it is known under the name ‘violetilla’. In this study, we report the occurrence of the cyclotide hypa A in H. parviflorus, together with a modified sample clean-up and capture procedure. We also propose a principle for uniform naming of new members of this growing family of peptides.
2. Results and discussion In our previous work on cyclotides from the Violaceae (Claeson et al., 1998; Go¨ransson et al., 1999), we have used a fractionation protocol originally developed for polypeptides in general from plant biomass (Claeson et al., 1998). Here we utilise the pronounced lipophilic nature of the cyclotides by exchanging two steps in the original protocol, gel filtration on Sephadex G-10 and solid phase extraction on RP-18 silica, for a simple solvent–solvent partitioning between water and butanol. The butanolic fraction is then directly amenable to high-performance chromatography techniques (i.e. RP–HPLC).
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00173-X
48
A.M. Broussalis et al. / Phytochemistry 58 (2001) 47–51
Table 1 The taxonomic distribution of known cyclotides Family
Taxa
Reference
Violaceae
Hybanthus parviflorus Leonia cymosa C. Martius Viola arvensis Murray V. odorata Linn. V. hederacea Labill.
This work (Hallock et al., 2000) (Go¨ransson et al., 1999; Claeson et al., 1998) (Craik et al., 1999) (Craik et al., 1999)
Rubiaceae
Chassalia parvifolia K. Schum. Oldenlandia affinis DC. Palicourea condensate Standl. Psychotria longipes Muell. Arg.
(Gustafson et al., 2000; Gustafson et al., 1994) (Craik et al., 1999; Gran, 1973a,b; Sletten and Gran, 1973) (Bokesch et al., 2001) (Witherup et al., 1994)
Cucurbitaceae
Momordica chinensis Hort.
(Hernandez et al., 2000)
Thus, in this investigation of H. parviflorus, the plant material was first defatted with CH2Cl2 and then extracted with EtOH:H2O 1:1. The acidified extract was filtered through polyamide to remove tannins before being partitioned between H2O and n-BuOH. The nBuOH extractives were subjected to repeated RP-HPLC to yield a homogeneous substance with an experimentally determined molecular mass of 3143 Da [M]. In comparison to the calculated theoretical mass given by the quantitative amino acid and Edman sequencing analyses (Table 2), a difference of 24 Da was observed. This agrees with previously isolated cyclotides, and is readily explained by the macrocyclic structure. Ring closure of the N- and C-terminals and the six cysteines engaged in three disulfide bridges corresponds to a mass loss of 24 Da. The peptide was then reduced and alkylated with 4vinylpyridine to yield the S-(ß-4-pyridylethyl) cysteine (PEC) derivative. The experimentally determined mass was 3780 Da [M], which agrees with the theoretical mass (3780 Da [M]) of a derivative with all six cysteines alkylated by 4-vinylpyridine. Subsequent digestion with endoproteinase Glu-C provided a single linear alkylated peptide (observed [M], 3798 Da; calculated [M], 3798 Da). The complete sequence of this derivative was then determined by automated Edman degradation and verified by MS2. The results are shown in Table 2 and in Figs. 1 and 2. Overall, this novel peptide exhibited a high degree of sequence similarity with the previously known cyclotides from Rubiaceae and Violaceae (Craik et al., 2001), and was 90% identical with the closest homologue, cycloviolacin O1, isolated from V. odorata. The disulfide arrangement in hypa A is most probably identical with the one reported from the NMR-structure of cycloviolacin O1 (Craik et al., 1999), i.e. C2–C17, C7–C22 and C15–C28. Moreover, the alignment placed the peptide in subfamily 1, the bracelet cyclotides, distinguished from the Moebius subfamily 2 by the lack of a cis-amide bond in a Trp-Pro sequence (Craik et al., 1999).
Fig. 1. The amino acid sequence of the cyclotide hypa A. The cleavage site of endoproteinase Glu-C, marked with an arrow, was chosen as an arbitrary starting point of the numbering of the amino acids (trypsin cleavage sites are marked with dashed arrows).
Cyclotides have hitherto been found in Leonia and Viola of the Violaceae family. This identification (the first) of a member of this substance class in Hybanthus supports chemotaxonomically the placing of H. parviflorus in Violaceae. The total number of known cyclotides now exceeds 40, and that figure will no doubt grow. With the principles for naming them having varied considerably, we propose that the trivial name should be constructed as an indicative and pronounceable acronym of the Latin binomial of the plant from which the cyclotide was first isolated, followed by a letter indicating the order of appearance. By these principles, this first cyclotide from Hybanthus parviflorus is named hypa A.
3. Experimental For HPLC, a Shimadzu LC-10 system was used, equipped with a SPD-M10Avp photodiode array detector. UV data were collected between 200 and 300 nm, 2504.6 (id) and 25010 (id) mm Rainin Dynamax (C18, 5 mm, 300 A˚) columns were used for analytical and
A.M. Broussalis et al. / Phytochemistry 58 (2001) 47–51
49
Table 2 The amino acid (aa) composition and molecular weight of hypa A. Residues from amino acid analysis are listed to the left, and the residues from sequencing, to the right Amino acid Asp/Asn (D/N) Thr (T) Ser (S) Glu/Gln (E/Q) Pro (P) Gly (G) Ala (A) Cys (C) Val (V) Ile (I) Leu (L) Tyr (Y) Lys (K) No aa:s Mr Calculated [M]c MS [M]d
Hypa A 2.0 1.9 2.0 1.0 2.0 2.1 2.0 5.0a 2.0 2.8 2.0 1.8 2.0 30
2N 2 2 1E 2 2 2 6b 2 3 2 2 2
3143 3143
a Half-cystine was determined as cysteic acid with a separate sample following oxidation with performic acid. b Cysteine, as (pyridylethyl)cysteine, following alkylation with 4vinylpyridine. c Calculated using average masses with the total sum from amino acid composition adjusted to the macrocyclic structure ( 18 Da) and three disulfide bridges ( 6 Da). d Determined by nanospray MS.
preparative RP–HPLC. For MS and MS2, a nanosprayion trap MS (Protana’s NanoES source mounted on a Finnigan LCQ MS) was used. All samples were sprayed in 50% MeOH, 1% HOAc, the capillary temperature was set to 230 C and the spray voltage to 0.8 kV. Average isotopic masses were used for all calculated molecular weights. 3.1. Plant material Aerial parts of H. parviflorus [syn. Ionidium glutinosum Ventetat, Viola parvifolia Roemer & Schultes (Ballard and Jorgensen, 1997)] were collected along the road No.12 and its intersection with Arroyo Feliciano, Department La Paz, Entre Rı´os Province, Argentina, during October 1998. A voucher specimen, identified by Dr. Juan de Dios Mun˜oz, is deposited at the herbarium of the Facultad de Ciencias Agrarias, Universidad Nacional de Entre Rı´os, Parana´, Argentina with the collection number Mun˜oz 1514 (ERA). After sun and oven drying at a temperature no more than 60 C, the plant material was ground to a fine powder. 3.2. Extraction and isolation Powdered aerial plant material (30.9 g) was extracted by maceration for 1 h with 300 ml of CH2Cl2 in a beaker mounted on a shaking table. This procedure was
Fig. 2. MS2-confirmation of the amino acid sequence after digestion with trypsin and endoproteinase Glu-C. Enzymatic peptides were isolated by RP-HPLC and analysed with nanospray-ion trap MS2 (35% CID; observed fragment masses are listed in the Experimental section). The two peptides sequenced here cover 28 out of 30 amino acids. The remaining dipeptide, N19-K20, was confirmed in MS2 fragmentation of a 30 amino acid linear peptide produced by partial digestion with trypsin (N19–K18, data not shown). (A) The fragmentation spectra of the ion 1052.5 [M+2H]2+. Assignment of the b (N-terminal fragments) and y series (C-terminal fragments) verifies 18 amino acids, S1 to K18. (B) Fragmentation of the ion 1183.3 [M+H]+, verifies the 10 amino acids before the cleavage point of GluC, V21 to E30 (bO is bH2O). The assignment of this peptide is not as straightforward as in (A), due to the lack of basic amino acids.
repeated 7 times, and the CH2Cl2-soluble extractives were discarded. The plant residue was dried overnight at room temperature and macerated 3 times in an analogous manner with 400 ml of EtOH:H2O 1:1. The combined alcoholic extracts were evaporated to dryness in vacuo, then tannins were removed by dissolving the extract in 2% AcOH and passing it through 31 g of polyamide gel (Polyamide 6S Riedel-de Hae¨n, Seelze, Germany) as described previously (Claeson et al., 1998). The yield was 4.5 g of lyophilized powder. A portion, 42 mg, of the tannin-free extract was dissolved
50
A.M. Broussalis et al. / Phytochemistry 58 (2001) 47–51
Acknowledgements
in H2O (10 ml) and partitioned 3 times against n-BuOH (10 ml). The butanolic phases were combined to yield 0.84 mg of extractives after removal of the solvent, which was then subjected to preparative RP-HPLC using a 25010 (i.d.) mm column eluted with a linear gradient from 25% CH3CN, 0.1% TFA to 60% CH3CN/0.1%TFA (v/v) over 30 min at a flow rate of 4 ml/min. For final purification by RP–HPLC, we used a 2504.6 (i.d.) mm column eluted with a gradient of 45– 48% organic modifier (CH3CH/i-PrOH, 6/4), 0.1% TFA, over 40 min at a flow rate of 1 ml/min. The yield of hypa A was 0.2 mg, as determined by quantitative amino acid analysis.
We thank Dr. Juan de Dios Mun˜oz at the herbarium of the Facultad de Ciencias Agrarias, Universidad Nacional de Entre Rı´os, Parana´ for identifying the voucher specimen, Prof. Ernst Oliw, Dept. of Pharmaceutical Biosciences, Uppsala University, for technical advice concerning LCQ MS, and Dr. A˚ke Engstro¨m in the Dept. of Medical Biochemistry and Microbiology, Uppsala University for assistance with Edman sequencing. We also thank the Swedish Institute, Stockholm, for a scholarship that enabled one of us (A.M.B.) to spend a sabbatical leave from the University of Buenos Aires at Uppsala University.
3.3. Determination of primary structure
References
Quantitative amino acid analysis was done at the Amino Acid Analysis Centre, Department of Biochemistry, Uppsala University. The peptide was hydrolysed for 24 h at 110 C with 6 N HCl containing 2 mg/ml phenol; the hydrolysates were analysed with an amino acid analyser using ninhydrin detection. For the sequence analysis, the peptide was reduced with dithioerythritol in 0.25 M Tris–HCl containing 1 mM EDTA and 6 M guanidine-HCl (pH 8.5; 24 C; 2 h). The reduced peptide was subsequently alkylated to its PEC derivative by adding 4-vinylpyridine to the solution (37 C; 1 h). Desalting and isolation of the alkylated peptide was done using gel filtration on a Superdex Peptide HR 10/30 column (Amersham Pharmacia Biotech, Uppsala, Sweden) in 40% CH3CN in 0.1% aqueous TFA. The cyclic PEC derivative was digested with endoproteinase Glu-C (sequencing grade, Promega Co., WI, USA) in 50 mM ammonium bicarbonate (pH 7.8; 37 C; 4 h). The linear peptide was isolated using gradient RPHPLC [2504.6 (i.d.) mm, H2O/CH3CN in 0.1% TFA], and its amino acid sequence was determined by automated Edman degradation using a protein sequencer. We then verified the sequence by MS2 analyses of endoproteinase Glu-C and tryptic fragments (sequencing grade modified trypsin, Promega Co., WI, USA; 50 mM NH4HCO3 pH 7.8; 37 C; 4 h) of the cyclic peptide alkylated with iodoacetamide. Experimental masses [M+H]+of assigned MS2-fragments (nomenclature according to Biemann, 1990): Fig. 2A, b-series: 1958.0 (b17), 1796.7 (b16), 1710.7 (b15), 1549.5 (b14), 1492.5 (b13), 1379.5 (b12), 1266.5 (b11), 1195.4 (b10), 1094.3 (b9), 981.1 (b8), 878.7 (b7), 720.3 (b6), 623.1 (b5), 510.0 (b4), 346.9 (b3), y-series: 1855.9 (y16), 1758.9 (y15), 1594.7 (y14), 1480.7 (y13), 1384.8 (y12), 1223.5 (y11), 1122.4 (y10), 1009.5 (y9), 908.4 (y8), 837.3 (y7), 724.1 (y6), 611.2 (y5); Fig. 2B, b-series: 1165.4 (b10), 1036.3 (b9), 965.3 (b8), 805.3 (b7), 707.1 (b6), y-series: 1084.3 (y9), 923.1 (y8), 760.2 (y7), 646.1 (y6), 589.5 (y5), 476.1 (y4).
Ballard, H.E., Jorgensen, P.M., 1997. A new name for an endemic Ecuadorian violet. Novon 7 (1), 13. Biemann, K., 1990. Nomenclature for peptide fragment ions. Method Enzymol. 193, 886–887. Bokesch, H.R., Pannell, L.K., Cochran, P.K., Sowder, R.C., McKee, T.C., Boyd, M.R., 2001. A novel anti-HIV macrocyclic peptide from Palicourea condensata. J. Nat. Prod. 64, 249–250. Claeson, P., Go¨ransson, U., Johansson, S., Luijendijk, T., Bohlin, L., 1998. Fractionation protocol for the isolation of polypeptides from plant biomass. J. Nat. Prod. 61, 77–81. Correa, M.N., 1988. Flora Patago´nica, Parte V Dicotiledones dialipe´talas. Coleccio´n Cientı´fica del INTA, Buenos Aires. Craik, D.J., Daly, N.L., Bond, T., Waine, C., 1999. Plant cyclotides: a unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif. J. Mol. Biol. 294, 1327–1336. Craik, D.J., Daly, N.L., Waine, C., 2001. The cystine knot motif in toxins and implications for drug design. Toxicon. 39, 43–60. Garcı´a Barriga, H., 1992. Flora Medicinal de Colombia, Bota´nica Me´dica, Tomo Segundo. Tercer Mundo Editores, Colombia. Go¨ransson, U., Luijendijk, T., Johansson, S., Bohlin, L., Claeson, P., 1999. Seven novel macrocyclic polypeptides from Viola arvensis. J. Nat. Prod. 62, 283–286. Gustafson, K.R., Walton, L.K., Sowder, R.C., Johnson, D.G., Pannell, L.K., Cardellina, J.H., Boyd, M.R., 2000. New circulin macrocyclic polypeptides from Chassalia parvifolia. J. Nat. Prod. 63, 176–178. Gustafson, K.R., Sowder, R.C., Henderson, L.E., Parsons, I.C., Kashman, Y., Cardellina, J.H., McMahon, J.B., Buckheit, R.W., Pannell, L.K., Boyd, M.R., 1994. Circulin A and circulin B — novel HIV-inhibitory macrocyclic peptides from the tropical tree Chassalia parvifolia. J. Am. Chem. Soc. 116, 9337–9338. Gran, L., 1973a. Oxytocic principles of Oldenlandia affinis. Lloydia 36, 174–178. Gran, L., 1973b. On the effect of a polypeptide isolated from ‘‘Kalatakalata’’ (Oldenlandia affinis DC) on the oestrogen dominated uterus. Acta Pharmacol. Toxicol. 33, 400–408. Hallock, Y.F., Sowder, R.C., Pannell, L.K., Hughes, C.B., Johnson, D.G., Gulakowski, R., Cardellina, J.H., Boyd, M.R., 2000. Cycloviolins A-D, anti-HIV macrocyclic peptides from Leonia cymosa. J. Org. Chem. 65, 124–128. Hernandez, J.F., Gagnon, J., Chiche, L., Nguyen, T.M., Andrieu, J.P., Heitz, A., Hong, T.T., Pham, T.T.C., Nguyen, D.L., 2000. Squash trypsin inhibitors from Momordica cochinchinensis exhibit an atypical macrocyclic structure. Biochemistry 39, 5722–5730. Mazorca, A., 1997. Vademecum de Malezas Medicinales de la Argentina, Indı´genas y Exo´ticas. Orientacio´n Gra´fica Editora SRL, Buenos Aires.
A.M. Broussalis et al. / Phytochemistry 58 (2001) 47–51 Murillo, A., 1889. Plantes me´dicinales du Chili. In: Roger, A. (Ed.), Exposition Universelle de Paris. Imprimerie de Lagny, Chernoviz, pp. 18. Rutter, R.A., 1990. Cata´logo de Plantas Utiles de la Amazonia Peruana. Ministerio de Educacio´n, Lima. Sletten, K., Gran, L., 1973. Some molecular properties of kalatapeptide B-1. Medd. Nor. Farm. Selsk. 35, 69–82. Soukup, J., 1986. Vocabulario de los Nombres Vulgares de la Flora Peruana y Cata´logo de los Ge´neros. Editorial Salesiana, Lima.
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Witherup, K.M., Bogusky, M.J., Anderson, P.S., Ramjit, H., Ransom, R.W., Wood, T., Sardana, M., 1994. Cyclopsychotride-A, a biologically active, 31-residue cyclic peptide isolated from Psychotria longipes. J. Nat. Prod. 57, 1619–1625. Zuloaga, F.O., Morrone, O., 1999. Cata´logo de las Plantas Vasculares de la Repu´blica Argentina II, Monographs in Systematic Botany from the Missouri Botanical Garden 74. Missouri Botanical Garden Press, St. Louis.
First cyclotide from Hybanthus (Violaceae) Adriana M. Broussalisa,1, Ulf Go¨ranssonb,1, Jorge D. Coussioa, Graciela Ferraroa, Virginia Martinoa, Per Claesonb,* a
Instituto de Quı´mica y Metabolismo del Fa´rmaco (IQUIMEFA) (UBA-CONICET), Ca´tedra de Farmacognosia, Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires, Argentina b Division of Pharmacognosy, Department of Medicinal Chemistry, Uppsala University, Biomedical Centre, Box 574, SE-751 23 Uppsala, Sweden Received 26 January 2001; received in revised form 23 March 2001
Abstract Hypa A, a novel macrocyclic polypeptide containing 30 amino acid residues, has been isolated from the n-butanol extract of the Argentine plant Hybanthus parviflorus. The sequence, cyclo-(SCVYIPCTITALLGCSCKNKVCYNGIPCAE), was determined by automated Edman degradation, quantitative amino acid analysis and nanospray MS/MS2. Three intramolecular disulfide bridges stabilize the cyclic peptide backbone of hypa A. Using these structural features to classify the peptide as a cyclotide, we extended the distribution of that substance class to a new genus, and now propose a uniform nomenclature for cyclotides. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Hybanthus parviflorus (Mutis ex L. F.) Baill.; Violaceae; Violetilla; Isolation; Primary structure; Cyclotides; Macrocyclic polypeptides; Hypa A; LCQ
1. Introduction The generic name ‘cyclotides’ (cyclic peptides) has been given to a rapidly emerging family of macrocyclic polypeptides, which are approximately 30 amino acid residues in length and are cyclized via the amide bonds of the peptide backbone (Craik et al., 1999). Their known taxonomic distribution has been limited to genera of Violaceae and Rubiaceae (Table 1). Recently, the characteristic cyclic cystine knot has also been found in peptides isolated from the family Cucurbitaceae (Hernandez et al., 2000). To further investigate the occurrence of cyclotides within Violaceae, we examined Hybanthus parviflorus (Mutis ex L. F.) Baill. H. parviflorus is a perennial shrub, widely distributed in the tropical and subtropical regions of America from Colombia to Argentina (Correa, 1988). In annual cultivations, it is a weed that grows on stubble, walls, rubble and wasteland (Zuloaga and Morrone, 1999). In several South American countries, including Colombia, Chile, * Corresponding author. Tel.: +46-18-471-44-79; fax: +46-18-50-9101. E-mail address: [email protected] (P. Claeson). 1 These authors contributed equally to this work.
Peru and Argentina, it is used as an emetic and purgative drug, and has substituted for ipecacuanha in medicine (Soukup, 1986; Murillo, 1889; Rutter, 1990; Garcı´a Barriga, 1992; Mazorca, 1997). In Argentina, it is known under the name ‘violetilla’. In this study, we report the occurrence of the cyclotide hypa A in H. parviflorus, together with a modified sample clean-up and capture procedure. We also propose a principle for uniform naming of new members of this growing family of peptides.
2. Results and discussion In our previous work on cyclotides from the Violaceae (Claeson et al., 1998; Go¨ransson et al., 1999), we have used a fractionation protocol originally developed for polypeptides in general from plant biomass (Claeson et al., 1998). Here we utilise the pronounced lipophilic nature of the cyclotides by exchanging two steps in the original protocol, gel filtration on Sephadex G-10 and solid phase extraction on RP-18 silica, for a simple solvent–solvent partitioning between water and butanol. The butanolic fraction is then directly amenable to high-performance chromatography techniques (i.e. RP–HPLC).
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00173-X
48
A.M. Broussalis et al. / Phytochemistry 58 (2001) 47–51
Table 1 The taxonomic distribution of known cyclotides Family
Taxa
Reference
Violaceae
Hybanthus parviflorus Leonia cymosa C. Martius Viola arvensis Murray V. odorata Linn. V. hederacea Labill.
This work (Hallock et al., 2000) (Go¨ransson et al., 1999; Claeson et al., 1998) (Craik et al., 1999) (Craik et al., 1999)
Rubiaceae
Chassalia parvifolia K. Schum. Oldenlandia affinis DC. Palicourea condensate Standl. Psychotria longipes Muell. Arg.
(Gustafson et al., 2000; Gustafson et al., 1994) (Craik et al., 1999; Gran, 1973a,b; Sletten and Gran, 1973) (Bokesch et al., 2001) (Witherup et al., 1994)
Cucurbitaceae
Momordica chinensis Hort.
(Hernandez et al., 2000)
Thus, in this investigation of H. parviflorus, the plant material was first defatted with CH2Cl2 and then extracted with EtOH:H2O 1:1. The acidified extract was filtered through polyamide to remove tannins before being partitioned between H2O and n-BuOH. The nBuOH extractives were subjected to repeated RP-HPLC to yield a homogeneous substance with an experimentally determined molecular mass of 3143 Da [M]. In comparison to the calculated theoretical mass given by the quantitative amino acid and Edman sequencing analyses (Table 2), a difference of 24 Da was observed. This agrees with previously isolated cyclotides, and is readily explained by the macrocyclic structure. Ring closure of the N- and C-terminals and the six cysteines engaged in three disulfide bridges corresponds to a mass loss of 24 Da. The peptide was then reduced and alkylated with 4vinylpyridine to yield the S-(ß-4-pyridylethyl) cysteine (PEC) derivative. The experimentally determined mass was 3780 Da [M], which agrees with the theoretical mass (3780 Da [M]) of a derivative with all six cysteines alkylated by 4-vinylpyridine. Subsequent digestion with endoproteinase Glu-C provided a single linear alkylated peptide (observed [M], 3798 Da; calculated [M], 3798 Da). The complete sequence of this derivative was then determined by automated Edman degradation and verified by MS2. The results are shown in Table 2 and in Figs. 1 and 2. Overall, this novel peptide exhibited a high degree of sequence similarity with the previously known cyclotides from Rubiaceae and Violaceae (Craik et al., 2001), and was 90% identical with the closest homologue, cycloviolacin O1, isolated from V. odorata. The disulfide arrangement in hypa A is most probably identical with the one reported from the NMR-structure of cycloviolacin O1 (Craik et al., 1999), i.e. C2–C17, C7–C22 and C15–C28. Moreover, the alignment placed the peptide in subfamily 1, the bracelet cyclotides, distinguished from the Moebius subfamily 2 by the lack of a cis-amide bond in a Trp-Pro sequence (Craik et al., 1999).
Fig. 1. The amino acid sequence of the cyclotide hypa A. The cleavage site of endoproteinase Glu-C, marked with an arrow, was chosen as an arbitrary starting point of the numbering of the amino acids (trypsin cleavage sites are marked with dashed arrows).
Cyclotides have hitherto been found in Leonia and Viola of the Violaceae family. This identification (the first) of a member of this substance class in Hybanthus supports chemotaxonomically the placing of H. parviflorus in Violaceae. The total number of known cyclotides now exceeds 40, and that figure will no doubt grow. With the principles for naming them having varied considerably, we propose that the trivial name should be constructed as an indicative and pronounceable acronym of the Latin binomial of the plant from which the cyclotide was first isolated, followed by a letter indicating the order of appearance. By these principles, this first cyclotide from Hybanthus parviflorus is named hypa A.
3. Experimental For HPLC, a Shimadzu LC-10 system was used, equipped with a SPD-M10Avp photodiode array detector. UV data were collected between 200 and 300 nm, 2504.6 (id) and 25010 (id) mm Rainin Dynamax (C18, 5 mm, 300 A˚) columns were used for analytical and
A.M. Broussalis et al. / Phytochemistry 58 (2001) 47–51
49
Table 2 The amino acid (aa) composition and molecular weight of hypa A. Residues from amino acid analysis are listed to the left, and the residues from sequencing, to the right Amino acid Asp/Asn (D/N) Thr (T) Ser (S) Glu/Gln (E/Q) Pro (P) Gly (G) Ala (A) Cys (C) Val (V) Ile (I) Leu (L) Tyr (Y) Lys (K) No aa:s Mr Calculated [M]c MS [M]d
Hypa A 2.0 1.9 2.0 1.0 2.0 2.1 2.0 5.0a 2.0 2.8 2.0 1.8 2.0 30
2N 2 2 1E 2 2 2 6b 2 3 2 2 2
3143 3143
a Half-cystine was determined as cysteic acid with a separate sample following oxidation with performic acid. b Cysteine, as (pyridylethyl)cysteine, following alkylation with 4vinylpyridine. c Calculated using average masses with the total sum from amino acid composition adjusted to the macrocyclic structure ( 18 Da) and three disulfide bridges ( 6 Da). d Determined by nanospray MS.
preparative RP–HPLC. For MS and MS2, a nanosprayion trap MS (Protana’s NanoES source mounted on a Finnigan LCQ MS) was used. All samples were sprayed in 50% MeOH, 1% HOAc, the capillary temperature was set to 230 C and the spray voltage to 0.8 kV. Average isotopic masses were used for all calculated molecular weights. 3.1. Plant material Aerial parts of H. parviflorus [syn. Ionidium glutinosum Ventetat, Viola parvifolia Roemer & Schultes (Ballard and Jorgensen, 1997)] were collected along the road No.12 and its intersection with Arroyo Feliciano, Department La Paz, Entre Rı´os Province, Argentina, during October 1998. A voucher specimen, identified by Dr. Juan de Dios Mun˜oz, is deposited at the herbarium of the Facultad de Ciencias Agrarias, Universidad Nacional de Entre Rı´os, Parana´, Argentina with the collection number Mun˜oz 1514 (ERA). After sun and oven drying at a temperature no more than 60 C, the plant material was ground to a fine powder. 3.2. Extraction and isolation Powdered aerial plant material (30.9 g) was extracted by maceration for 1 h with 300 ml of CH2Cl2 in a beaker mounted on a shaking table. This procedure was
Fig. 2. MS2-confirmation of the amino acid sequence after digestion with trypsin and endoproteinase Glu-C. Enzymatic peptides were isolated by RP-HPLC and analysed with nanospray-ion trap MS2 (35% CID; observed fragment masses are listed in the Experimental section). The two peptides sequenced here cover 28 out of 30 amino acids. The remaining dipeptide, N19-K20, was confirmed in MS2 fragmentation of a 30 amino acid linear peptide produced by partial digestion with trypsin (N19–K18, data not shown). (A) The fragmentation spectra of the ion 1052.5 [M+2H]2+. Assignment of the b (N-terminal fragments) and y series (C-terminal fragments) verifies 18 amino acids, S1 to K18. (B) Fragmentation of the ion 1183.3 [M+H]+, verifies the 10 amino acids before the cleavage point of GluC, V21 to E30 (bO is bH2O). The assignment of this peptide is not as straightforward as in (A), due to the lack of basic amino acids.
repeated 7 times, and the CH2Cl2-soluble extractives were discarded. The plant residue was dried overnight at room temperature and macerated 3 times in an analogous manner with 400 ml of EtOH:H2O 1:1. The combined alcoholic extracts were evaporated to dryness in vacuo, then tannins were removed by dissolving the extract in 2% AcOH and passing it through 31 g of polyamide gel (Polyamide 6S Riedel-de Hae¨n, Seelze, Germany) as described previously (Claeson et al., 1998). The yield was 4.5 g of lyophilized powder. A portion, 42 mg, of the tannin-free extract was dissolved
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A.M. Broussalis et al. / Phytochemistry 58 (2001) 47–51
Acknowledgements
in H2O (10 ml) and partitioned 3 times against n-BuOH (10 ml). The butanolic phases were combined to yield 0.84 mg of extractives after removal of the solvent, which was then subjected to preparative RP-HPLC using a 25010 (i.d.) mm column eluted with a linear gradient from 25% CH3CN, 0.1% TFA to 60% CH3CN/0.1%TFA (v/v) over 30 min at a flow rate of 4 ml/min. For final purification by RP–HPLC, we used a 2504.6 (i.d.) mm column eluted with a gradient of 45– 48% organic modifier (CH3CH/i-PrOH, 6/4), 0.1% TFA, over 40 min at a flow rate of 1 ml/min. The yield of hypa A was 0.2 mg, as determined by quantitative amino acid analysis.
We thank Dr. Juan de Dios Mun˜oz at the herbarium of the Facultad de Ciencias Agrarias, Universidad Nacional de Entre Rı´os, Parana´ for identifying the voucher specimen, Prof. Ernst Oliw, Dept. of Pharmaceutical Biosciences, Uppsala University, for technical advice concerning LCQ MS, and Dr. A˚ke Engstro¨m in the Dept. of Medical Biochemistry and Microbiology, Uppsala University for assistance with Edman sequencing. We also thank the Swedish Institute, Stockholm, for a scholarship that enabled one of us (A.M.B.) to spend a sabbatical leave from the University of Buenos Aires at Uppsala University.
3.3. Determination of primary structure
References
Quantitative amino acid analysis was done at the Amino Acid Analysis Centre, Department of Biochemistry, Uppsala University. The peptide was hydrolysed for 24 h at 110 C with 6 N HCl containing 2 mg/ml phenol; the hydrolysates were analysed with an amino acid analyser using ninhydrin detection. For the sequence analysis, the peptide was reduced with dithioerythritol in 0.25 M Tris–HCl containing 1 mM EDTA and 6 M guanidine-HCl (pH 8.5; 24 C; 2 h). The reduced peptide was subsequently alkylated to its PEC derivative by adding 4-vinylpyridine to the solution (37 C; 1 h). Desalting and isolation of the alkylated peptide was done using gel filtration on a Superdex Peptide HR 10/30 column (Amersham Pharmacia Biotech, Uppsala, Sweden) in 40% CH3CN in 0.1% aqueous TFA. The cyclic PEC derivative was digested with endoproteinase Glu-C (sequencing grade, Promega Co., WI, USA) in 50 mM ammonium bicarbonate (pH 7.8; 37 C; 4 h). The linear peptide was isolated using gradient RPHPLC [2504.6 (i.d.) mm, H2O/CH3CN in 0.1% TFA], and its amino acid sequence was determined by automated Edman degradation using a protein sequencer. We then verified the sequence by MS2 analyses of endoproteinase Glu-C and tryptic fragments (sequencing grade modified trypsin, Promega Co., WI, USA; 50 mM NH4HCO3 pH 7.8; 37 C; 4 h) of the cyclic peptide alkylated with iodoacetamide. Experimental masses [M+H]+of assigned MS2-fragments (nomenclature according to Biemann, 1990): Fig. 2A, b-series: 1958.0 (b17), 1796.7 (b16), 1710.7 (b15), 1549.5 (b14), 1492.5 (b13), 1379.5 (b12), 1266.5 (b11), 1195.4 (b10), 1094.3 (b9), 981.1 (b8), 878.7 (b7), 720.3 (b6), 623.1 (b5), 510.0 (b4), 346.9 (b3), y-series: 1855.9 (y16), 1758.9 (y15), 1594.7 (y14), 1480.7 (y13), 1384.8 (y12), 1223.5 (y11), 1122.4 (y10), 1009.5 (y9), 908.4 (y8), 837.3 (y7), 724.1 (y6), 611.2 (y5); Fig. 2B, b-series: 1165.4 (b10), 1036.3 (b9), 965.3 (b8), 805.3 (b7), 707.1 (b6), y-series: 1084.3 (y9), 923.1 (y8), 760.2 (y7), 646.1 (y6), 589.5 (y5), 476.1 (y4).
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